Fusion polypeptides capable of activating receptors

ABSTRACT

A fusion polypeptide comprising (A) x -M-(A′) y , wherein A and A′ are each polypeptides capable of binding a target receptor. The fusion polypeptides of the invention form multimeric proteins which activate the target receptor. A and A′ may be each be an antibody or fragment derived from an antibody specific for a target receptor, such as the same or different ScFv fragments, and/or a ligand or ligand fragment or derivative capable of binding the target protein, M is a multimerizing component, and X and Y are independently a number between 1-10.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional 60/536,968 filed 16 Jan. 2004, which application is hereinspecifically incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to multimeric fusion proteins capable ofactivating a target receptor, methods of producing such fusionpolypeptides, and methods for treating, diagnosing, or monitoringdiseases or conditions in which activation of the target receptor isdesired.

2. Description of Related Art

The clustering of soluble Eph ligand domains to create multimers capableof activating their cognate receptors is described in U.S. Pat. No.5,747,033. U.S. Pat. No. 6,319,499 recites a method of activating anerythropoietin receptor with an antibody.

BRIEF SUMMARY OF THE INVENTION

The present invention provides multimeric fusion polypeptides capable ofactivating a target receptor requiring multimerization to be activated.The polypeptides of the invention are useful for treating conditions inwhich activation of a target receptor is desirable, as well as having avariety of in vitro and in vivo diagnostic and prognostic uses. Thepolypeptides of the invention may be monospecific or bispecifictetramers exhibiting improved capacity to activate a target receptorrelative to, for example, a target-specific antibody or the naturalligand.

Accordingly, in a first aspect the invention provides an isolatednucleic acid molecule which encodes a fusion polypeptide(A)_(x)-M-(A′)_(y), wherein A is a polypeptide specific for a targetreceptor, M is a multimerizing component, A′ is a polypeptide specificfor the same target receptor as A, and X and Y are independently anumber between 1-10.

In a first embodiment, A and A′ are antibodies or antibody fragmentsspecific to the target receptor, and are the same antibody or antibodyfragment specific to a target receptor. In another embodiment, A and A′are different antibodies or antibody fragments specific to the sametarget receptor. Preferably, A and A′ are single chain Fv (ScFv)fragments. When the fusion polypeptide is intended as a humantherapeutic, the invention encompasses humanized antibody or antibodyfragments.

In a second embodiment, A and A′ are ligands or ligand fragmentsspecific for the same target receptor. In a more specific embodiment, Aand A′ are the same or are different ligands or ligand fragmentsspecific to the same target receptor.

In a third embodiment, A is an antibody or antibody fragment specific tothe target receptor, and A′ is a ligand or ligand fragment specific tothe same target receptor. In preferred embodiments, A is an antibody orantibody fragment to a Tie receptor (Tie-1 or Tie-2), and A′ is thefibrinogen domain of a Tie receptor.

In specific embodiments, M is a multimerizing component whichmultimerizes with a multimerizing component on another fusionpolypeptide to form a multimer of the fusion polypeptides. In apreferred embodiment, M is the Fc domain of IgG or the heavy chain ofIgG. The Fc domain of IgG may be selected from the isotypes IgG1, IgG2,IgG3, and IgG4, as well as any allotype within each isotype group.

In a second aspect the invention provides a fusion polypeptidecomprising (A)_(x)-M-(A′)_(y), wherein A is a polypeptide specific for atarget receptor, M is a multimerizing component, A′ is a polypeptidespecific for the same target receptor as A, and X and Y areindependently a number between 1-10.

In a first embodiment, A and A′ are antibodies or antibody fragmentsspecific to the target receptor, and are the same antibody or antibodyfragment specific to a target receptor. In another embodiment, A and A′are different antibodies or antibody fragments specific to the sametarget receptor. Preferably, A and A′ are single chain Fv (ScFv)fragments.

In a second embodiment, A and A′ are ligands or ligand fragmentsspecific for the same target receptor. In a more specific embodiment, Aand A′ are different ligands or ligand fragments specific to the sametarget receptor. In another specific embodiment, A and A′ are the sameligand or ligand fragment.

In a third embodiment, A is an antibody or antibody fragment specific tothe target receptor, and A′ is a ligand or ligand fragment specific tothe same target receptor.

In a third aspect, the invention provides an activating dimeric fusionpolypeptide comprising two fusion polypeptides of the invention, e.g., adimer formed from two polypeptides of (A)_(x)-M-(A′)_(y) as definedabove. The activating dimers of the invention are capable of binding toand clustering four or more receptors, leading to receptor activation,as compared with the ability of an antibody to cluster no more than tworeceptors.

In one embodiment, the components of the fusion polypeptides of theinvention are connected directly to each other. In other embodiments, aspacer sequence may be included between one or more components, whichmay comprise one or more molecules, such as amino acids. For example, aspacer sequence may include one or more amino acids naturally connectedto a domain-containing component. A spacer sequence may also include asequence used to enhance expression of the fusion polypeptide, providerestriction sites, allow component domains to form optimal tertiary andquaternary structures and/or to enhance the interaction of a componentwith its target receptor. In one embodiment, the fusion polypeptide ofthe invention comprises one or more peptide sequences between one ormore components which is(are) between 1-25 amino acids. Furtherembodiments may include a signal sequence at the beginning oramino-terminus of an fusion polypeptide of the invention. Such a signalsequence may be native to the cell, recombinant, or synthetic.

The components of the fusion polypeptide of the invention may bearranged in a variety of configurations. For example, described from thebeginning or amino-terminus of the fusion polypeptide,(A)_(x)-M-(A′)_(y), (A)_(x)-(A′)_(y)-M, M-(A)_(x)-(A′)_(y),(A′)_(Y)-M-(A)_(X), (A′)_(Y)-(A)_(X)-M, M-(A′)_(Y)-(A)_(X),(A)_(x)-M-(A′)_(y), (A)_(x)-(A′)_(y)-M, M-(A)_(x)-(A′)_(y), etc.,wherein X=1-10 and Y=1-10. In an even more specific embodiment, X=1, andY=1 or X=2 and Y=2.

In a fourth aspect, the invention features a vector comprising a nucleicacid sequence of the invention. The invention further features anexpression vector comprising a nucleic acid of the invention, whereinthe nucleic acid molecule is operably linked to an expression controlsequence. Also provided is a host-vector system for the production ofthe fusion polypeptides of the invention which comprises the expressionvector of the invention which has been introduced into a host cell ororganism, including, but not limited to, transgenic animals, suitablefor expression of the fusion polypeptides.

In a fifth aspect, the invention features a method of producing a fusionpolypeptide of the invention, comprising culturing a host celltransfected with a vector comprising a nucleic acid sequence of theinvention, under conditions suitable for expression of the polypeptidefrom the host cell, and recovering the fusion polypeptide so produced.

In a sixth aspect, the invention features therapeutic methods for thetreatment of a target receptor-related disease or condition, comprisingadministering a therapeutically effective amount of an activating dimerof the invention to a subject in need thereof, wherein the targetreceptor is activated, and the disease or condition is ameliorated orinhibitited.

Accordingly, in a seventh aspect, the invention features pharmaceuticalcompositions comprising an activating dimer of the invention with apharmaceutically acceptable carrier. Such pharmaceutical compositionsmay comprise dimeric proteins or nucleic acids which encode them.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

As used herein, the term “target receptor-related condition or disease”generally encompasses a condition of a mammalian host, particularly ahuman host, which is associated with a particular target receptor. Thus,treating a target receptor-related condition will encompass thetreatment of a mammal, in particular, a human, who has symptomsreflective of decreased target receptor activation, or who is expectedto have such decreased levels in response to a disease, condition ortreatment regimen. Treating an target receptor-related condition ordisease encompasses the treatment of a human subject wherein enhancingthe activation of a target receptor with an activating dimer of theinvention results in amelioration of an undesirable symptom resultingfrom the target receptor-related condition or disease. As used herein,an “target receptor-related condition” also includes a condition inwhich it is desirable to alter, either transiently, or long-term,activation of a particular target receptor.

Target Receptors

Examples of target receptors are members of the Eph family (e.g. EphA1,EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3,EphB4, EphB5, EphB6), Tie receptors (e.g. Tie-1 or Tie-2). Suitableligands or fragments thereof include the soluble domain of an ephrin(e.g. ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1,ephrin-B2, ephrin-B3), and the fibrinogen domain of an angiopoietin(e.g. angiopoietin-1 (ang-1), ang-2, ang-3, ang-4).

Suitable target receptors are receptors that are activated whenmultimerized. This class of receptors includes, but is not limited to,those that possess an integral kinase domain. Within this class ofintegral kinase receptors are those that form homodimers, or clusters ofthe same receptor, such as Tie-1, Tie-2, EGFR, FGFR, the Trk family andthe Eph family of receptors, and those that form heterodimers, orclusters, such as the VEGF receptors VEGFR1, VEGFR2, the PDGF receptorsPDGFRα and PDGFRβ, and the TGF-β family receptors. Suitable targetreceptors also include, but are not limited to, the class of receptorswith associated kinases. These receptors include those that formhomodimers, or clusters, such as the growth hormone receptor, EPOR andthe G-CSF receptor CD114, and those that form heterodimers, or clusters,such as the GM-CSF receptors GMRα and GMRβ

Target Receptor-Specific Antibodies and Ligands

In specific embodiments, the activating dimers of the invention compriseone or more immunoglobulin binding domains isolated from antibodiesgenerated against a selected target receptor. The term “immunoglobulin”or “antibody” as used herein refers to a mammalian, including human,polypeptide comprising a framework region form an immunoglobulin gene orfragments thereof that specifically binds and recognizes an antigen,which, in the case of the present invention, is a target receptor orportion thereof. If the intended activating dimer will be uses as ahuman therapeutic, immunoglobulin binding regions should be derived fromthe corresponding human immunoglobulins or be a humanizedimmunoglobulin. The human immunoglobulin genes or gene fragments includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constantregions, as well as the myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.Within each IgG class, there are different isotypes e.g., IgG₁, IgG₂,etc.). Typically, the antigen-binding region of an antibody will be themost critical in determining specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit of human IgG,comprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one light chain (about 25 kD)and one heavy chain (about 50-70 kD). The N-terminus of each chaindefines a variable region of about 100-110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins, or as a number ofwell-characterized fragments produced by digestion with variouspeptidases, e.g., F(ab)′₂, Fab′, etc. Thus, the terms immunoglobulin orantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies, or those synthesizedde novo using recombinant DNA methodologies (e.g., single chain Fv)(ScFv) or those identified using phase display libraries (see, forexample, McCafferty et al. (1990) Nature 348:552-554). In addition, thetarget receptor-binding domain component of the fusion polypeptides ofthe invention include the variable regions of the heavy (V_(H)) or thelight (V_(L)) chains of immunoglobulins, as well as targetreceptor-binding portions thereof. Methods for producing such variableregions are described in Reiter, et al. (1999) J. Mol. Biol.290:685-698.

Methods for preparing antibodies are known to the art. See, for example,Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988)Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.). The genes encoding the heavy and light chains of anantibody of interest can be cloned from a cell, e.g., the genes encodinga monoclonal antibody can be cloned from a hybridoma and used to producea recombinant monoclonal antibody. Gene libraries encoding heavy andlight chains of monoclonal antibodies can also be made from hybridoma orplasma cells. Random combinations of the heavy and light chain geneproducts generate a large pool of antibodies with different antigenicspecificity. Techniques for the production of single chain orrecombinant antibodies (U.S. Pat. No. 4,946,778; U.S. Pat. No.4,816,567) can be adapted to produce antibodies used in the fusionpolypeptides, activating dimers and methods of the instant invention.Also, transgenic mice, or other organisms such as other mammals, may beused to express human or humanized antibodies. Alternatively, phagedisplay technology can be used to identify antibodies, antibodyfragments, such as variable domains, and heteromeric Fab fragments thatspecifically bind to selected antigens. Phage display is of particularvalue to isolate weakly binding antibodies or fragments thereof fromunimmunized animals which, when combined with other weak binders inaccordance with the invention described herein, create strongly bindingactivating dimers.

Screening and selection of preferred immunoglobulins (antibodies) can beconducted by a variety of methods known to the art. Initial screeningfor the presence of monoclonal antibodies specific to an target receptormay be conducted through the use of ELISA-based methods or phagedisplay, for example. A secondary screen is preferably conducted toidentify and select a desired monoclonal antibody for use inconstruction of the fusion polypeptides of the invention. Secondaryscreening may be conducted with any suitable method known to the art.

Nucleic Acid Construction and Expression

Individual components of the fusion polypeptides of the invention may beproduced from nucleic acids molecules using molecular biological methodsknown to the art. Nucleic acid molecules are inserted into a vector thatis able to express the fusion polypeptides when introduced into anappropriate host cell. Appropriate host cells include, but are notlimited to, bacterial, yeast, insect, and mammalian cells. Any of themethods known to one skilled in the art for the insertion of DNAfragments into a vector may be used to construct expression vectorsencoding the fusion polypeptides of the invention under control oftranscriptional/translational control signals. These methods may includein vitro recombinant DNA and synthetic techniques and in vivorecombinations (See Sambrook et al. Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory; Current Protocols in MolecularBiology, Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience,NY).

Expression of the nucleic acid molecules of the invention may beregulated by a second nucleic acid sequence so that the molecule isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression of the nucleic acid molecules of the invention maybe controlled by any promoter/enhancer element known in the art.

Immunoglobulin-derived components. The nucleic acid constructs includeregions which encode binding domains derived from an anti-targetreceptor antibodies. In general, such binding domains will be derivedfrom V_(H) or V_(L) chain variable regions. After identification andselection of antibodies exhibiting the desired binding characteristics,the variable regions of the heavy chains and/or light chains of eachantibody is isolated, amplified, cloned and sequenced. Modifications maybe made to the V_(H) and V_(L) nucleotide sequences, including additionsof nucleotide sequences encoding amino acids and/or carrying restrictionsites, deletions of nucleotide sequences encoding amino acids, orsubstitutions of nucleotide sequences encoding amino acids.

The invention encompasses antibodies or antibody fragments which arehumanized or chimeric. “Humanized” or chimeric forms of non-human (e.g.,murine) antibodies are immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or otherantigen-binding subsequences of antibodies) that contain minimalsequences required for antigen binding derived from non-humanimmunoglobulin. They have the same or similar binding specificity andaffinity as a mouse or other nonhuman antibody that provides thestarting material for construction of a chimeric or humanized antibody.Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalantibody may be joined to human constant (C) segments, such as IgG1 andIgG4. Human isotype IgG1 is preferred. A typical chimeric antibody isthus a hybrid protein consisting of the V or antigen-binding domain froma mouse antibody and the C or effector domain from a human antibody.Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions (CDR regions) substantially from amouse antibody, (referred to as the donor immunoglobulin). See, Queen etal., Proc. Natl. Acad Sci. USA 86:10029-10033 (1989) and WO 90/07861,U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539.The constant region(s), if present, are also substantially or entirelyfrom a human immunoglobulin. The human variable domains are usuallychosen from human antibodies whose framework sequences exhibit a highdegree of sequence identity with the murine variable region domains fromwhich the CDRs were derived. The heavy and light chain variable regionframework residues can be derived from the same or different humanantibody sequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See WO 92/22653. Certain amino acids from thehuman variable region framework residues are selected for substitutionbased on their possible influence on CDR conformation and/or binding toantigen. Investigation of such possible influences is by modeling,examination of the characteristics of the amino acids at particularlocations, or empirical observation of the effects of substitution ormutagenesis of particular amino acids. For example, when an amino aciddiffers between a murine variable region framework residue and aselected human variable region framework residue, the human frameworkamino acid should usually be substituted by the equivalent frameworkamino acid from the mouse antibody when it is reasonably expected thatthe amino acid: (1) noncovalently binds antigen directly; (2) isadjacent to a CDR region; (3) otherwise interacts with a CDR region(e.g. is within about 6 A of a CDR region), or (4) participates in theV_(L)-V_(H) interface. Other candidates for substitution are acceptorhuman framework amino acids that are unusual for a human immunoglobulinat that position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. Othercandidates for substitution are acceptor human framework amino acidsthat are unusual for a human immunoglobulin at that position. Thevariable region frameworks of humanized immunoglobulins usually show atleast 85% sequence identity to a human variable region frameworksequence or consensus of such sequences.

Fully human antibodies may be made by any method known to the art. Forexample, U.S. Pat. No. 6,596,541 describes a method of generating fullyhuman antibodies. Briefly, initially a transgenic animal such as a mouseis generated that produces hybrid antibodies containing human variableregions (VDJ/VJ) and mouse constant regions. This is accomplished by adirect, in situ replacement of the mouse variable region (VDJ/VJ) geneswith their human counterparts. The mouse is then exposed to humanantigen, or an immunogenic fragment thereof. The resultant hybridimmunoglobulin loci will undergo the natural process of rearrangementsduring B-cell development to produce hybrid antibodies having thedesired specificity. The antibody of the invention is selected asdescribed above. Subsequently, fully-human antibodies are made byreplacing the mouse constant regions with the desired humancounterparts. Fully human antibodies can also be isolated from mice orother transgenic animals such as cows that express human transgenes orminichromosomes for the heavy and light chain loci. (Green (1999) JImmunol Methods. 231:11-23 and Ishida et al (2002) Cloning Stem Cells.4:91-102) Fully human antibodies can also be isolated from humans towhom the protein has been administered. Fully human antibodies can alsobe isolated from immune compromised mice whose immune systems have beenregenerated by engraftment with human stem cells, splenocytes, orperipheral blood cells (Chamat et al (1999) J Infect Dis. 180:268-77).To enhance the immune response to the protein of interest one canknockout the gene encoding the protein of interest in thehuman-antibody-transgenic animal.

Receptor-binding domains. In accordance with the invention, the nucleicacid constructs include components which encode binding domains derivedfrom target receptor ligands. After identification of a ligand's targetreceptor-binding domain exhibiting desired binding characteristics, thenucleic acid that encodes such domain is used in the nucleic acidconstructs. Such nucleic acids may be modified, including additions ofnucleotide sequences encoding amino acids and/or carrying restrictionsites, deletions of nucleotide sequences encoding amino acids, orsubstitutions of nucleotide sequences encoding amino acids.

The nucleic acid constructs of the invention are inserted into anexpression vector or viral vector by methods known to the art, whereinthe nucleic acid molecule is operatively linked to an expression controlsequence. Also provided is a host-vector system for the production ofthe fusion polypeptides and activating dimers of the invention, whichcomprises the expression vector of the invention, which has beenintroduced into a suitable host cell. The suitable host cell may be abacterial cell such as E. coli, a yeast cell, such as Pichia pastoris,an insect cell, such as Spodoptera frugiperda, or a mammalian cell, suchas a COS, CHO, 293, BHK or NS0 cell.

The invention further encompasses methods for producing the activatingdimers of the invention by growing cells transformed with an expressionvector under conditions permitting production of the fusion polypeptidesand recovery of the activating dimers formed from the fusionpolypeptides. Cells may also be transduced with a recombinant viruscomprising the nucleic acid construct of the invention.

The activating dimers may be purified by any technique, which allows forthe subsequent formation of a stable dimer. For example, and not by wayof limitation, the activating dimers may be recovered from cells eitheras soluble polypeptides or as inclusion bodies, from which they may beextracted quantitatively by 8M guanidinium hydrochloride and dialysis.In order to further purify the activating dimers, conventional ionexchange chromatography, hydrophobic interaction chromatography, reversephase chromatography or gel filtration may be used. The activatingdimers may also be recovered from conditioned media following secretionfrom eukaryotic or prokaryotic cells.

Screening and Detection Methods

The activating dimers of the invention may also be used in in vitro orin vivo screening methods where it is desirable to detect and/or measuretarget receptor levels. Screening methods are well known to the art andinclude cell-free, cell-based, and animal assays. In vitro assays can beeither solid state or soluble target receptor detection may be achievedin a number of ways known to the art, including the use of a label ordetectable group capable of identifying an activating dimer which isbound to an target receptor. Detectable labels are well developed in thefield of immunoassays and may generally be used in conjunction withassays using the activating dimer of the invention.

Therapeutic Methods

The ability of the activating dimers of the invention to exhibit highaffinity binding for their receptors makes them therapeutically usefulfor efficiently activating their receptors. Thus, it certain instancesit may be to increase the effect of endogenous ligands for targetreceptors, such as, for example, the ephrins. For example, in the areaof nervous system trauma, certain conditions may benefit from anincrease in ephrin responsiveness. It may therefore be beneficial toincrease the binding affinity of an ephrin in patients suffering fromsuch conditions through the use of the compositions described herein.

The invention herein further provides for the development of anactivating dimer described herein as a therapeutic for the treatment ofpatients suffering from disorders involving cells, tissues or organswhich express the Tie-2 receptor. Such molecules may be used in a methodof treatment of the human or animal body, or in a method of diagnosis.

The target receptor known as Tie-2 receptor has been identified inassociation with endothelial cells and, as was previously demonstrated,blocking of agonists of the receptor such as Tie-2 ligand 1 (Ang1) hasbeen shown to prevent vascularization. Accordingly, activating dimers ofthe invention wherein the target receptor is Tie-2 may be useful for theinduction of vascularization in diseases or disorders where suchvascularization is indicated. Such diseases or disorders would includewound healing, ischemia and diabetes. The ligands may be tested inanimal models and used therapeutically as described for other agents,such as vascular endothelial growth factor (VEGF), another endothelialcell-specific factor that is angiogenic. Ferrara et al. U.S. Pat. No.5,332,671 issued Jul. 26, 1994. Ferrara et al. describe in vitro and invivo studies that may be used to demonstrate the effect of an angiogenicfactor in enhancing blood flow to ischemic myocardium, enhancing woundhealing, and in other therapeutic settings wherein neoangiogenesis isdesired. According to the invention, such a Tie-2 specific activatingdimer may be used alone or in combination with one or more additionalpharmaceutically active compounds such as, for example, VEGF or basicfibroblast growth factor (bFGF).

Methods of Administration

Methods known in the art for the therapeutic delivery of agents such asproteins or nucleic acids can be used for the therapeutic delivery of anactivating dimer or a nucleic acid encoding an activating dimer of theinvention for activating target receptors in a subject, e.g., cellulartransfection, gene therapy, direct administration with a deliveryvehicle or pharmaceutically acceptable carrier, indirect delivery byproviding recombinant cells comprising a nucleic acid encoding anactivating dimer of the invention.

Various delivery systems are known and can be used to administer theactivating dimer of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction can beenteral or parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary,intranasal, intraocular, epidural, and oral routes. The compounds may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. Pulmonary administration canalso be employed, e.g., by use of an inhaler or nebulizer, andformulation with an aerosolizing agent.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising an activating dimer of the invention and a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. (See, forexample, “Remington's Pharmaceutical Sciences” by E. W. Martin.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Where necessary, thecomposition may also include a solubilizing agent and a local anestheticsuch as lidocaine to ease pain at the site of the injection. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The active agents of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with at least one activating dimer of theinvention. The kits of the invention may be used in any applicablemethod, including, for example, diagnostically. Optionally associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects (a)approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

Transgenic Animals

The invention includes transgenic non-human animals expressing a fusionpolypeptide of the invention. A transgenic animal can be produced byintroducing nucleic acid into the male pronuclei of a fertilized oocyte,e.g., by microinjection, retroviral infection, and allowing the oocyteto develop in a pseudopregnant female foster animal. Any of theregulatory or other sequences useful in expression vectors can form partof the transgenic sequence. A tissue-specific regulatory sequence(s) canbe operably linked to the transgene to direct expression of thetransgene to particular cells. A transgenic non-human animal expressingan fusion polypeptide of the invention is useful in a variety ofapplications, including as a means of producing such fusion proteins.Further, the transgene may be placed under the control of an induciblepromoter such that expression of the fusion polypeptide may becontrolled by, for example, administration of a small molecule.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Production of Anti-Tie-2 Hybridomas

Five 8-weeks old Balb/c mice were first immunized with purified humanTie-2-Fc (hTie2-Fc); each mouse was injected subcutaneously with 200 μlemulsion containing 100 μg purified hTie2-Fc protein and 100 μl Freund'scomplete adjuvant. Fifteen days after the primary injection, each mousereceived subcutaneous injection of 200 μl emulsion containing 100 μgpurified hTie2-Fc in 100 μl PBS and 100 μl Freund's incomplete adjuvant.This injection was repeated for the five mice seven days later. Onemouse was used for generation of hybridomas against hTie-2. Each of thefour remaining mice were given subcutaneous injections of 200 μlemulsion each containing 100 μg purified rat Tie-2-Fc (rTie2-Fc) in 100μl PBS and 100 μl Freund's incomplete adjuvant six months after theprimary injection of hTie2-Fc. Eleven days later, the immune response ofthe mice to rTie2-Fc was boosted by subcutaneous injection of 200 μl ofemulsion containing 100 μg purified rTie2-Fc in 100 μl PBS and 100 μlFreund's incomplete adjuvant for each mouse. Mouse sera were collectedfrom tail veins three days after the injection, then the antibody titersagainst rTie2-Fc were determined by ELISA. The two mice with the highesttiters were given a final boost by tail vein injection of 100 μgpurified rTie2-Fc in 100 μl PBS. The mice were sacrificed three dayslater and their spleen cells were collected for fusion with Sp2/0-Ag14cells.

To generate hybridomas, mouse spleen cells were fused with Sp2/0-Ag14myeloma cells using polyethylene glycol (PEG). Briefly, after thespleens were aseptically removed from the mice, one tip of each spleenwas cut open and spleen cells collected. The spleen cells were washedtwice with D-MEM and cell numbers were counted using a hemocytometer.2×10⁸ spleen cells were combined with 3×10⁷ Sp2/0-Ag14 cells that werein log growth stage. The cell mix was washed with 30 mls D-MEM. 1 ml 50%PEG at 37° C. was slowly added to the cell pellet while stirring. D-MEMwas added to the mix to bring the volume to 10 mls. The cells were spundown at 400×g for 10 minutes. After removal of supernatant, the cellswere gently resuspended in 20 mls growth medium containing 60% D-MEMwith 4.5 g/L glucose, 20% FCS, 10% NCTC109 medium, 10% hybridoma cloningfactor, 1 mM oxaloacetate, 2 mM glutamine, 0.2 units/ml insulin, and 3μM glycine. The cells were transferred to two T225 flasks, eachcontaining 100 mls of the growth medium and were put into a tissueculture incubator. On the next day, 1×HAT was added to the medium toselect against the myeloma cells that were not fused. Nine days afterthe fusion, the cultures were replenished with fresh medium. Human IgGwas added to the cultures at 1 mg/ml. On the tenth day after the fusion,2.6×10⁷ fused cells were stained sequentially with 1 μg/mlbiotin-rTie2-Fc for one hour and 2.5 μg/ml phycoerythrin (PE)-conjugatedstreptavidin for 45 minutes in growth medium at room temperature. As acontrol, 1×10⁶ fused cells were stained with 2.5 μg/ml PE-streptavidinfor 45 minutes at room temperature. The cells were washed with 10 ml PBSafter each stain. After staining, the cells were resuspended in PBS with0.1% FCS and were analyzed by flow cytometry on a MoFlo (Cytomation). Apopulation of cells (4% total cells) stained with both biotin rTie2-Fcand PE-streptavidin exhibited fluorescence higher than the unstainedcells and the cells stained with PE-streptavidin alone. The cells inthis 4% gate were cloned by sorting single cells into two 96-well platescontaining 200 μl growth medium per well. The cells were cultured for 10days before splitting into two sets of 96-well plates. Cells in one setof plate were processed for RT-PCR of mouse IgG heavy chain variableregion following by sequencing. The clones were grouped into 14 bins,with members of each bin having identical sequence in their heavy chainvariable region. Conditioned medium of hybridoma cells in each bin wastested for its ability to stimulate phosphorylation of rTie-2 incultured rat aortic endothelial cells (RAECs).

Antibodies from two hybridomas, B2 and A12A, were chosen for furtherstudy because they were active in phosphorylation of Tie-2 in RAECs, anddid not compete for binding to rTie-2 as determined by BIAcore analysis.In addition, these antibodies did not block binding of derivatives ofangiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), the natural ligands ofTie-2.

Example 2 Construction of ScFvs (B2 and A12A)

Generally, antibody variable regions from hybridomas expressingantibodies specific for rTie-2 were cloned by first determining the DNAsequence of RT-PCR products using primers specific for mouse antibodyvariable regions, then using specific primers based on the determinedsequence in order to amplify DNA fragments encoding ScFvs. The ScFv DNAfragments were cloned such that they could be cassette exchanged withmultiple plasmids to yield all combinations of activating dimers. Forexample, one amplified ScFv fragment could be fused to a signal sequenceat the N-terminus and to a coding sequence for the IgG Fc domain at theC-terminus, or it could be fused to the C-terminus of an IgG Fc codingsequence such that the 3′ end of the ScFv coding sequence contained atranslation stop codon.

The B2 hybridoma was found to express an antibody capable of inducingphosphorylation of the Tie-2 receptor in RAECs. Total RNA was isolatedfrom this hybridoma using the promega SV96 Total RNA Isolation System(Promega) and variable heavy cDNA was synthesized using the QiagenOne-Step RT-PCR system (Qiagen) with heavy chain primers from the Ratnerprimer set (Wang et al. (2000) J. Immunol. Methods 233:167) thatincluded equimolar amounts of the 5′ primers (SEQ ID NO:1-7) and the 3′primer (SEQ ID NO:8). Similarly, the light chain variable regions wereamplified from cDNA using equimolar amounts of the light chain-specificprimers (SEQ ID NO:9 and 10). The amplified variable region fragmentswere cloned into the pCR2.1-TOPO vector (Invitrogen) and the DNAsequences were determined. Based on the determined variable regionsequences for the B2 antibody, the variable heavy sequence was PCRamplified using the pCR2.1-TOPO cloned variable region as template andan equimolar mix of 5′ and 3′ primers (SEQ ID NO: 19 and SEQ ID NO: 20).The variable light sequence was PCR amplified using a similar strategy.The pCR2.1-TOPO cloned variable region was used as template and anequimolar mix of 5′ and 3′ primers (SEQ ID NO:21 and SEQ ID NO:22). Thevariable regions were joined by a (G4S)3 linker; ScFv genes wereassembled and PCR amplified using an equimolar mix of the above specificvariable heavy and variable light PCR products and an equimolar mix of5′ B2 heavy primer (SEQ ID NO:19) and the 3′ light primer (SEQ IDNO:22). PCR product was cloned into Invitrogen pCR2.1-TOPO (Invitrogen)to yield pRG1039. The sequence was confirmed before sub-cloning the 744bp AscI/SrfI to fuse the ScFv gene to the N-terminus of a DNA encodingthe human IgG1 Fc fragment (hFc), or the 753 bp AscI/NotI restrictionfragments to fuse the same ScFv to the C-terminus of a DNA encoding hFc.

The A12A hybridoma was also found to express an antibody capable ofinducing phosphorylation of the Tie-2 receptor in RAECs. Total RNA wasisolated from this hybridoma using the Quick Prep mRNA purification kit(Amersham Pharmacia Biotech) and variable heavy cDNA was synthesizedusing the Qiagen One-Step RT-PCR system, with equimolar amounts ofprimers from the from the Wright primer set (Morrison et al. (1995)Antibody Engineering, second edition, Borrebaeck, C. K. A. editor267-293) that included the 5′ heavy chain primers (SEQ ID NO: 11-13) andthe 3′ primer (SEQ ID NO:8). Similarly, the light chain variable regionswere amplified from cDNA with equimolar amounts of the 5′ heavy chainprimers (SEQ ID NO: 14-18) and the 3′ primer (SEQ ID NO:10). Theamplified variable region fragments were cloned into the pCR2.1-TOPOvector (Invitrogen) and the DNA sequences were determined.

Based on the determined variable region sequences for the A12A antibody,the variable heavy sequence was PCR amplified using the pCR2.1-TOPOcloned variable region as template and an equimolar mix of 5′ and 3′primers (SEQ ID NO:23 and SEQ ID NO:24). The variable light sequence wasPCR amplified using a similar strategy. The pCR2.1-TOPO cloned variableregion was used as template and an equimolar mix of 5′ and 3′ primers(SEQ ID NO:25 and SEQ ID NO:26). The variable regions were joined by a(G₄S)₃ linker; ScFv genes were assembled and PCR amplified using anequimolar mix of the above specific variable heavy and variable lightPCR products and an equimolar mix of 5′ A12A heavy primer (SEQ ID NO:23)and the 3′ light primer (SEQ ID NO:26). PCR product was cloned intoInvitrogen pCR2.1-TOPO to yield pRG1090. The sequence was confirmedbefore sub-cloning the 747 bp AscI/SrfI to fuse the ScFv gene to theN-terminus of a DNA encoding the hFc fragment.

Example 3 Construction of Monospecific and Bispecific Activating Dimers

The general scheme for constructing both monspecific and bispecifictetravalent activating dimers was based on the ability of either the B2or A12A ScFv genes to be inserted between the murine ROR1 signalsequence (SEQ ID NO:27) and the gene encoding hFc (nucleotides 85 to 765of GenBank accession # X70421) when cut with one set of restrictionenzymes, or after the hFc gene if cut with a different set of enzymes.This design of the ScFv genes allowed the exchange of ScFv cassettesamong plasmids to obtain different combinations of ScFv and hFc usingstandard known methods. All constructs have an optional three amino acidlinker (spacer) between the cleavage site of the signal peptide and thestart of the ScFv gene, resulting from engineering a restriction siteonto the 5′ end of the ScFv genes. Similarly, fusion to the aminoterminus of the hFc gene was facilitated by a three amino acid sequence(Gly-Pro-Gly), and fusion to the carboxy terminus of the hFc gene wasfacilitated by an eight amino acid sequence consisting of the residuesGly₄-Ser-Gly-Ala-Pro (SEQ ID NO:32) As a consequence of the terminalrestriction site linkers on the ScFv genes, all constructs that have acarboxy terminal ScFv end with the amino acids Gly-Pro-Gly.

Two types of svFc-based chimeric molecules were constructed to assessthe ability of ScFv-based molecules to activate the rTie-2 receptor. Onetype of molecule used a single ScFv fused to both the N-terminus and theC-terminus of hFc, the consequence of which was a monospecifictetravalent molecule capable of binding rTie-2. This molecule wasexpected to be capable of simultaneously binding four rTie-2 molecules.The plasmid pTE586 encodes the gene for ScFv_(B2)-Fc-ScFv_(B2) (SEQ IDNO: 29) whose secretion is directed by the mROR1 signal peptide. Theexpression of ScFv_(B2)-Fc-ScFv_(B2) in pTE586 was directed by theCMV-MIE promoter when transfected into CHO cells. This protein waseasily purified by Protein A-Sepaharose affinity chromatography.

Construction of an ScFv-Fc-ScFv molecule wherein the two ScFv domainsare derived from two different non-competing anti-rTie-2 antibodieswould yield a molecule capable of clustering more than four receptors,in contrast to the ScFv_(B2)-Fc-ScFv_(B2) described above, which cancluster only four receptors. It was determined by BIAcore analysis thatthe binding of the B2 antibody did not block binding of A12A to rTie-2,and A12A binding first did not block binding of B2. Consequently, ScFvmolecules made from these antibodies should be capable of clusteringmore than four receptors. To construct a bispecific tetravalentScFv-based molecule, the ScFv_(A12A) gene was used in combination withthe ScFv_(B2) gene to yield ScFv_(A12A)-Fc-ScFv_(B2) (SEQ ID NO: 28).The plasmid pTE585 encodes the gene for ScFv_(A12A)-Fc-ScFv_(B2) and hasthe mROR1 signal peptide and CMV-MIE promoter when transfected into CHOcells. Both ScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) wereexpressed in CHO cells, and purified by Protein A-Sepharose affinitychromatography.

Example 4 Assays

Antibodies to rTie-2, and chimeric molecules related to theseantibodies, were evaluated for their ability to induce phosphorylationof Tie-2 in cultured rat aortic endothelial cells. Confluent RAECs,between passage 3 and 6 (Vec Technologies), were grown in MCDB-131 media(Vec Technologies) on 0.2% gelatin coated T-75 flasks. Cells werestarved for 2 hrs. in serum-free DME-Hi glucose medium (IrvineScientific) prior to incubation at 37° C. for 5 min. in 1.5 mlserum-free DME-Hi glucose medium with 0.1% BSA and the challengemolecule. The challenge medium was then removed and cells were lysed in20 mM Tris, pH 7.6, 150 mM NaCl, 50 mM NaF, 1 mM Na orthovanadate, 5 mMbenzamidine, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,with 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF. Tie-2 wasimmunoprecipitated by incubating the lysates at 4° C. for 16 hrs. with 5μg anti-Tie-2 mouse monoclonal antibody KP-m33, 10 μg biotinylatedanti-mouse IgG (Jackson Laboratories), and 100 μl of neutravidin beads(Pierce). Beads were collected by centrifugation, washed 3 times withRIPA buffer, and bound proteins were eluted with 40 μl of 5× Laemmlibuffer with 10% B-mercaptoethanol by heating at 100° C. for 5 min. AfterSDS-gel electrophoresis on a 4-12% Tris/glycine polyacrylamide gel(Novex), proteins were transferred to PVDF membranes and probed withmouse anti-phophotyrosine monoclonal antibody 4G10 (Upstate) thendetected using goat anti-mouse IgG-HRP conjugate (Pierce) followed byECL reagent (Amersham). The ability to induce Tie-2 phosphorylation inRAECs was determined for each activating dimer. Activity was evaluatedby comparison to the level of stimulation obtained with FD1-Fc-FD1(BA1)—a chimeric protein shown to be as active as Ang1 in binding andactivation Tie-2 (Davis et al. (2003) Nature Struct. Biol. 10:38-44)(FD1 or FD2=human fibrinogen domain of Ang1 or Ang2, respectively).Maximum stimulation (ECmax) of Tie-2 in RAECs was observed when BA1 wasused at about 0.5 to 1.0 μg/ml, and phosphorylation levels in mocktreated cells were low. Similarly, the ECmax of ScFv_(B2)-Fc-ScFv_(B2),ScFv_(A12A)-Fc-ScFv_(B2), ScFv_(B2)-Fc-FD1, and ScFv_(B2)-Fc-FD2 wereabout 0.5 to 1.0 μg/ml. In all cases, the ScFv-based molecules werecapable of inducing a higher phosphorylation signal than observed forthe related native antibodies isolated from hybridoma conditioned media.

Purified ScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) werecharacterized for their ability to bind rTie-2 and inducephosphorylation. Binding to rTie-2 was determined by BIAcore analysis.Both the monospecific and the dispecific activating dimers were found tohave significantly higher affinity for rTie-2 than FD1-Fc-FD1. Inaddition, both ScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) wereable to stimulate phosphorylation of rTie-2 in RAECs comparable toFD1-Fc-FD1.

Example 5 Construction of ScFv/Ligand Activating Dimers

Bispecific tetravalent molecules were constructed to include both Tie-2specific ScFv and FD1 or FD2. The chimeric molecules were made by fusingthe gene encoding ScFv_(B2) to the N-terminus of hFc and the geneencoding Ang1 FD (Phe283 to Phe498 of GenBank accession # Q15389) orAng2 FD (Phe281 to Phe496 of GenBank accession # O15123) to theC-terminus. Plasmid pTE514 encodes the gene for ScFv_(B2)-Fc-FD1 (SEQ IDNO: 30) and contained the mROR1 signal peptide and CMV-MIE promoter.Plasmid pTE614 encodes the gene for ScFv_(B2)-Fc-FD2 (SEQ ID NO: 31) andcontained the mROR1 signal peptide and CMV-MIE promoter. Similar toScFv_(B2)-Fc-ScFv_(B2) and ScFv_(A12A)-Fc-ScFv_(B2) the proteinsexpressed from pTE514 and pTE614 had a Gly-Ala-Pro linker between themROR1 signal peptide and the ScFv_(B2), a Gly-Pro-Gly linker between theN-terminal ScFv_(B2) and hFc and a Gly₄-Ser-Gly-Ala-Pro linker (SEQ IDNO:32) between the C-terminus of hFc and the N-terminus of the Ang FDs.Both ScFv_(B2)-Fc-FD1 and ScFv_(B2)-Fc-FD2 were expressed and purifiedas described above.

Purified ScFv_(B2)-Fc-FD1 and ScFv_(B2)-Fc-FD2 were characterized fortheir ability to bind rTie-2 and induce phosphorylation as described inabove. As determined by BIAcore analysis, the chimeric activating dimerScFv_(B2)-Fc-RDI was found to have significantly higher affinity forrTie-2 (0.04 nM) than FD1-Fc-FD1 (2 nM). Moreover, bothScFvB_(B2)-Fc-FD1 and ScFv_(B2)-Fc-FD2 were able to stimulatephosphorylation of rTie-2 in RAECs comparable to FD1-Fc-FD1.

Example 6 Construction of Fully Human Activating Dimers

Bispecific tetravalent molecules are formed from dimerized fusionconstructs of the invention which include either two ScFvs derived fromhuman antibodies specific for hTie-2 or one ScFv derived from a humanantibody specific for hTie-2 and human FD1 or FD2. Human ScFvs specificfor hTie-2 are obtained by methods known to the art and as describedabove. In one embodiment, human ScFvs are obtained recombinantly asdescribed in Reiter et al. (1999) J. Mol. Biol. 290:685-698 andGilliland et al. (1996) Tissue Antigens 47(1):1-20.

Example 7 Construction of ScFvs (1-1F11 and 2-1G3)

Anti-rTie-1 hybridomas were produced following the procedures describedabove for the production of anti-rTie-2 hybridomas. Briefly, mice wereimmunized three times with purified rat Tie-1-Fc protein and Freund'sadjuvant. Spleen cells from the mouse with the highest anti-Tie-1antibody titer were fused with Sp2/0-Ag14 myeloma cells usingpolyethylene glycol (PEG). After fusion, the cells were cultured in twoT225 flasks. HAT was added to the cultures on the next day. Nine daysafter the fusion, the cultures were replenished with fresh medium. HumanIgG was added to the cultures at 1 mg/ml. On the tenth day after thefusion, the HAT-resistant cells were stained sequentially with 1 μg/mlbiotin-rat Tie-1-Fc for one hour and 2.5 μg/ml phycoerythrin(PE)-conjugated streptavidin for 45 minutes in growth medium at roomtemperature. After staining, the cells were analyzed by flow cytometery.Cells that bound rTie1-Fc were cloned by sorting single cells into96-well plates. The 96-well plate cultures were split into two sets tendays after sorting. RT-PCR of mouse IgG heavy chain variable regionfollowed by sequencing were performed on one set of the 96-well platecultures. Clones with unique IgG heavy chain variable region sequenceswere identified and expanded for the production of anti-rTie-1antibodies. Antibodies were tested for binding rTie-1 protein and twoclones, 1-1F11 and 1-2G3, were chosen for more detailed study.

The 1 -1F11 hybridoma was found to express an antibody capable ofinducing phosphorylation of the Tie-1 receptor in RAECs. Messenger RNAwas isolated and variable heavy cDNA synthesized as described above withheavy chain primers from the Wright primer set (Morrison et al. (1995)Antibody Engineering, second edition, Borrebaeck, C. K. A. editor267-293) that included the 5′ heavy chain primers (SEQ ID NO:35-37) andthe 3′ primer (SEQ ID NO:33). Similarly, the light chain variableregions were amplified from cDNA with equimolar amounts of the 5′ lightchain primers (SEQ ID NO:38-41) and the 3′ primer (SEQ ID NO:34). Theamplified variable region fragments were cloned into the pCR2.1-TOPOvector (Invitrogen) and DNA sequences determined. Based on thedetermined variable region sequences for the 1-1F11 antibody, thevariable heavy sequence was PCR amplified using the pCR2.1-TOPO clonedvariable region as template and an equimolar mix of 5′ and 3′ primers(SEQ ID NO:42 and SEQ ID NO:43). The variable light sequence was PCRamplified using a similar strategy. The pCR2.1-TOPO cloned variableregion was used as template and an equimolar mix of 5′ and 3′ primers(SEQ ID NO:44 and SEQ ID NO:45). The variable regions were joined by a(G₄S)₃ linker; ScFv genes were assembled and PCR amplified using anequimolar mix of the above specific variable heavy and variable lightPCR products and an equimolar mix of 5′ heavy primer (SEQ ID NO:42) andthe 3′ light primer (SEQ ID NO:45). PCR product was cloned intoInvitrogen pCR2.1-TOPO (Invitrogen) to yield pRG1192. The sequence wasconfirmed before sub-cloning the 747 bp AscI/SrfI to fuse the ScFv geneto the N-terminus of a DNA encoding the human IgG1 Fc fragment (hFc), orthe 756 bp AscI/NotI restriction fragments to fuse the same ScFv to theC-terminus of a DNA encoding hFc.

The 2-1G3 hybridoma was also found to express an antibody capable ofinducing phosphorylation of the Tie-1 receptor in RAECs. Messenger RNAwas isolated and variable heavy cDNA synthesized as described above withequimolar amounts of primers from the Wright primer set (Morrison et al.(1995) supra) that included the 5′ heavy chain primers (SEQ ID NO:35-37)and the 3′ primer (SEQ ID NO:33). Similarly, the light chain variableregions were amplified from cDNA with equimolar amounts of the 5′ heavychain primers (SEQ ID NO:38-41) and the 3′ primer (SEQ ID NO:34). Theamplified variable region fragments were cloned into the pCR2.1-TOPOvector (Invitrogen) and the DNA sequences were determined.

Based on the determined variable region sequences for the 2-1G3antibody, the variable heavy sequence was PCR amplified using thepCR2.1-TOPO cloned variable region as template and an equimolar mix of5′ and 3′ primers (SEQ ID NO:46 and SEQ ID NO:47). The variable lightsequence was PCR amplified using a similar strategy. The pCR2.1-TOPOcloned variable region was used as template and an equimolar mix of 5′and 3′ primers (SEQ ID NO:48 and SEQ ID NO:49). The variable regionswere joined by a (G4S)3 linker; ScFv genes were assembled and PCRamplified using an equimolar mix of the above specific variable heavyand variable light PCR products and an equimolar mix of 5′ 2-1G3 heavyprimer (SEQ ID NO:46) and the 3′ light primer (SEQ ID NO:49). PCRproduct was cloned into Invitrogen pCR2.1-TOPO (Invitrogen) to yieldpRG1198. The sequence was confirmed before sub-cloning the 738 bpAscI/SrfI to fuse the ScFv gene to the N-terminus of a DNA encoding thehFc fragment or the 747 bp AscI/NotI restriction fragments to fuse thesame ScFv to the C-terminus of a DNA encoding hFc.

Example 8 Construction of Monospecific and Bispecific Activating Dimers

Two types of ScFv-based chimeric molecules were constructed to assessthe ability of ScFv-based molecules to activate the rTie-1 receptor. Onetype of molecule used a single ScFv fused to both the N-terminus and theC-terminus of hFc, the consequence of which was a monospecifictetravalent molecule capable of binding rTie-1. This molecule should becapable of simultaneously binding four rTie-1 molecules. The plasmidpTE778 encodes the gene for ScFv_(1-1F11)-Fc-ScFv_(1-1F11) (SEQ IDNO:50) and contains the mROR1 signal peptide and CMV-MIE promoter. Theprotein was expressed and purified as described above.

Construction of an ScFv-Fc-ScFv molecule where the two ScFv domains arederived from two different non-competing anti-rTie-1 antibodies isexpected to yield a molecule capable of clustering more than fourreceptors, in contrast to the ScFv_(1-1F11)-Fc-ScFv_(1-1F11) describedabove, which can cluster only four receptors. It was determined byBIAcore analysis that the binding of the 1-1F11 antibody did not blockbinding of 2-1G3 to rTie-1, and 1-1F11 binding first did not blockbinding of 2-1G3. Consequently, ScFv molecules made from theseantibodies should be capable of clustering more than four receptors. Toconstruct a bispecific tetravalent ScFv-based molecule, the ScFv_(2-1G3)gene was used in combination with the ScFv_(1-1F11) gene to yieldScFv_(2-1G3)-Fc-ScFv_(1-1F11) (SEQ ID NO:51). Both constructs wereexpressed and purified as described above.

1. A nucleic acid encoding a fusion protein, wherein the fusion proteinis selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:50, and SEQ ID NO:51.
 2. An expressionvector encoding the fusion protein of claim
 1. 3. An isolated host cellcomprising the expression vector of claim
 2. 4. The nucleic acid ofclaim 1, encoding the fusion protein of SEQ ID NO:28.
 5. The nucleicacid of claim 1, encoding the fusion protein of SEQ ID NO:29.
 6. Thenucleic acid of claim 1, encoding the fusion protein of SEQ ID NO:30. 7.The nucleic acid of claim 1, encoding the fusion protein of SEQ IDNO:31.
 8. The nucleic acid of claim 1, encoding the fusion protein ofSEQ ID NO:50.
 9. The nucleic acid of claim 1, encoding the fusionprotein of SEQ ID NO:51.